Quantitative Assessment of Plasmodium Falciparum PNAS PLUS Sexual Development Reveals Potent Transmission- Blocking Activity by Methylene Blue

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Quantitative assessment of Plasmodium falciparum PNAS PLUS sexual development reveals potent transmission- blocking activity by methylene blue

Sophie H. Adjalleya,1, Geoffrey L. Johnstona,b, Tao Lic, Richard T. Eastmana,2, Eric H. Eklanda, Abraham G. Eappenc, Adam Richmanc, B. Kim Lee Simc, Marcus C. S. Leea, Stephen L. Hoffmanc, and David A. Fidocka,d,3

aDepartment of Microbiology and Immunology, Columbia University College of Physicians and Surgeons, New York, NY 10032; bSchool of International and Public Affairs, Columbia University, New York, NY 10027; cSanaria, Inc., Rockville, MD 20850; and dDivision of Infectious Diseases, Department of Medicine, Columbia University College of Physicians and Surgeons, New York, NY 10032

Edited* by Thomas E. Wellems, National Institutes of Health, Bethesda, MD, and approved October 5, 2011 (received for review July 25, 2011) Clinical studies and mathematical models predict that, to achieve the course of infection, allowing gametocytes to mature and malaria elimination, combination therapies will need to incorpo- continue the transmission cycle (4, 5). rate drugs that block the transmission of Plasmodium falciparum Most of the available information on the impact of existing sexual stage parasites to mosquito vectors. Efforts to measure the antimalarials on gametocyte development has been gathered activity of existing antimalarials on intraerythrocytic sexual stage from field-based clinical trials, and in vitro studies remain sparse gametocytes and identify transmission-blocking agents have, until (6). Activity against immature gametocytes has been observed now, been hindered by a lack of quantitative assays. Here, we with a number of drugs that target metabolic pathways in asexual report an experimental system using P. falciparum lines that stably blood stage parasites. These drugs include chloroquine and express gametocyte-specific GFP-luciferase reporters, which en- quinine, which interfere with heme detoxification, and atova- able the assessment of dose- and time-dependent drug action on quone, which targets mitochondrial electron transport (6, 7). gametocyte maturation and transmission. These studies reveal ac- Artemisinins have also been observed to inhibit immature sexual tivity of the first-line antimalarial dihydroartemisinin and the part- stages and reduce gametocyte carriage in infected human hosts ner drugs lumefantrine and pyronaridine against early gametocyte (8, 9). The experimental design of many of the studies, however, MICROBIOLOGY stages, along with moderate inhibition of mature gametocyte has often made it difficult to distinguish between direct game- transmission to Anopheles mosquitoes. The other partner agents tocytocidal activity and the antiasexual properties that reduce the monodesethyl-amodiaquine and piperaquine showed activity only pool of parasites available to initiate gametocytogenesis. Earlier against immature gametocytes. Our data also identify methylene in vitro evaluations of drug gametocytocidal activity typically blue as a potent inhibitor of gametocyte development across relied on laborious, subjective microscopic observations of par- all stages. This thiazine dye almost fully abolishes P. falciparum asite morphology to assess cell viability (6). Recent advances transmission to mosquitoes at concentrations readily achievable in include the use of hydroethidine or alamarBlue as fluorescent humans, highlighting the potential of this chemical class to reduce markers of metabolic activity (10, 11) or GFP reporter lines to the spread of malaria. assess commitment to gametocytogenesis (12, 13), notably after antimalarial drug treatment of asexual forms (10). These meth- artemisinin-based combination therapies | transfection ods, however, have yet to be exploited to assess dose-dependent drug effects over time or define rates of action with purified, ith ∼225 million individuals infected and an estimated mature gametocyte populations. Also, these studies have not W780,000 deaths annually—largely among African children— measured drug-mediated inhibition of P. falciparum transmission Plasmodium falciparum malaria remains one of the world’s most to Anopheles, a technically challenging endeavor. devastating diseases. Encouragingly, in recent years, the global Here, we have engineered a set of P. falciparum lines ex- fi adoption of highly effective artemisinin-based combination pressing stably integrated gametocyte-speci c GFP-luciferase fi therapies (ACTs) as first-line treatments and the distribution of reporters, and we have developed assays to speci cally monitor fi long-lasting insecticide-treated bednets have helped reduce the parasite sexual development throughout the ve stages of game- prevalence of malaria in many endemic settings (1). This recent tocytogenesis and determine the kinetics of gametocytocidal success contributed to the launch in 2007 of a new campaign action of antimalarial agents. Luciferase-based assays permitted of malaria eradication (2). Clinical reports and mathematical quantitative assessment of the effects of key primary and partner models show that additional reductions in the incidence of dis- components of ACTs on P. falciparum gametocyte maturation. ease can be achieved with interventions combining vector control Select compounds were then further investigated for their ability approaches and treatments that not only cure patients but also decrease transmission (3). Current antimalarials reduce P. falciparum malaria-associated morbidity and mortality by targeting the pathogenic asexual Author contributions: S.H.A., T.L., A.R., M.C.S.L., and D.A.F. designed research; S.H.A., T.L., R.T.E., A.G.E., and M.C.S.L. performed research; S.H.A., G.L.J., E.H.E., A.R., B.K.L.S., S.L.H., blood stages within infected erythrocytes. This activity indirectly and D.A.F. analyzed data; and S.H.A., G.L.J., E.H.E., M.C.S.L., S.L.H., and D.A.F. wrote impacts parasite transmission to its Anopheles mosquito vector the paper. by limiting the number of asexual forms that can differentiate The authors declare no conflict of interest. into mature intraerythrocytic gametocytes. Nevertheless, patients *This Direct Submission article had a prearranged editor. cleared of asexual parasites can still transmit surviving game- 1Present address: Genome Biology Unit, European Molecular Biology Laboratory, 69117 tocytes. Ideal combination therapies would, thus, target both the Heidelberg, Germany. rapidly replicating asexual stages and the less metabolically ac- 2Present address: Laboratory of Malaria and Vector Research, National Institute of Allergy tive, nonreplicating mature gametocytes. This ideal combination and Infectious Diseases, National Institutes of Health, Rockville, MD 20852. is of particular relevance in highly endemic regions, where 3To whom correspondence should be addressed. E-mail: [email protected]. P. falciparum infections are often asymptomatic and thus, go This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. untreated and where clinical symptoms tend to develop late in 1073/pnas.1112037108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1112037108 PNAS Early Edition | 1of10 Downloaded by guest on September 30, 2021 to inhibit gametocyte transmission to Anopheles vectors. Our plasmids and signal heterogeneity between parasites. Here, results show that ACT compounds in clinical use are generally transgene integration was applied to the NF54 strain, which active against immature gametocytes. We also observed partial produces highly infectious gametocytes and is being used in activity of dihydroartemisinin (DHA) against late stage game- human malaria vaccine trials (16). Our integration strategy was tocytes, consistent with a reduction in the number of oocysts in designed to eliminate the selectable marker that might otherwise mosquitoes fed on DHA-treated gametocytes. Exquisite potency interfere with antimalarial modes of action. was observed against early and mature gametocytes with the Bxb1 integrase catalyzes homology-directed recombination synthetic dye methylene blue (MB), which also almost fully between a short attP site and a target attB site. NF54 parasites blocked transmission. These results provide the foundation to containing an attB site were generated by transforming asexual identify transmission-blocking agents that can be integrated into blood stages with the pCC-cg6-attB plasmid, which contains an curative combination therapies. attB site cloned between sequences homologous to the cg6 gene along with the human dhfr-positive selectable marker and the Results cdup-negative marker (Fig. 1A). After selection for human dhfr + Engineering of Unmarked NF54attB Recombinant Parasites Amenable (using the antifolate WR99210), PCR was used to detect attB to Site-Specific Recombination. To produce P. falciparum reporter integrants that resulted from single cross-over recombination on lines expressing GFP-luciferase under the control of gametocyte- the 3′ side of cg6. Integration was confirmed by Southern blot specific promoters, we used Bxb1 mycobacteriophage integrase- hybridization (Fig. 1B), and clones were obtained by limiting mediated recombination to deliver transgenes into the P. falci- dilution. After removal of WR99210, the negative selection parum genome (14). Prior strategies have used transgenes on agent 5-fluorocytosine was applied to select for intralocus re- episomally replicating plasmids (13, 15), where the absence of combination between duplicated cg6 5′ sequences, leading to selection during gametocyte development can lead to the loss of excision of cdup and its flanking human dhfr marker (Fig. 1A).

A pGEM-3Z B S/N 4.9kb pCC-cg6-attB episomes

cdup hdhfr cg6 5’attBcg6 3’ bulk culture 2 bulk culture 1 + + kb empty pcc-cg6-attB NF54 wild-type attB S EE S cg6 3’ recombination attB NF54 wild-type 8.0 cg6 7.0 S S E N S 6.0 8.2kb 5.0

WR99210 selection 4.0

attB+ integrants (marked attB locus) S/E S/N S E N C cg6 cdup hdhfr cg6 5’attB cg6 3’ attB attB S EE S 6.3kb 2.7kb 6.6kb 4.9kb recombinant 1 recombinant 1 recombinant 2 recombinant 2 + + + + cg6 5’ recombination attB NF54 wild-type unmarked NF54 unmarked NF54 attB attB NF54 wild-type kb attB

5-fluorocytosine selection 8.0 7.0

NF54attB (unmarked attB locus) 5.0

4.0 cg6 5’attB cg6 3’ S E N 3.0 S 3.3kb

Fig. 1. Integration of an unmarked attB site for site-specific recombination in P. falciparum NF54 parasites. (A) Integration of the plasmid pCC-cg6-attB into the cg6 gene and subsequent intralocus recombination leading to removal of the selectable markers from the recombinant locus. This plasmid expresses human dihydrofolate reductase (hdhfr) and Saccharomyces cerevisiae cytosine deaminase–uracyl phosphotransferase (cdup) for positive and negative selection, respectively (58). cg6 5′ and 3′ coding sequences were placed on either side of an attB site to permit single cross-over homologous recombination between the pCC-cg6-attB plasmid and the endogenous cg6 gene. Single cross-overs were identified between the cg6 3′ homologous regions, leading to plasmid integration into the genome. Selection with 5-fluorocytosine, which is activated into a toxic compound by CDUP, was used to obtain parasites that have lost the hdhfr and cdup selectable markers. This selection was achieved through intralocus recombination between the duplicated cg6 5′ sequences, leading to the formation of a single unmarked attB locus. The black rectangle illustrates the 0.4-kb cg6 probe used for Southern blot analysis, and restriction digest fragment sizes are indicated. (B) Southern blot analysis confirming pCC-cg6-attB plasmid integration into NF54 parasites. DNAs were digested with SalI/ NruI (S/N) and hybridized with the cg6 probe. Bulk cultures contained a mixture of integrants that yielded band sizes of 6.6 and 4.9 kb (characteristic of plasmid integration in the 3′ region of cg6) as well as episomal transfectants that showed the 4.9-kb plasmid band and the 8.2-kb nonrecombinant cg6 locus. (C) Southern blot analysis confirming unmarking of attB+ parasites. DNAs were digested with SalI/EcoRV (S/E) or S/N and hybridized with the cg6 probe. This process produced bands of 6.8 and 8.2 kb, respectively, in NF54 wild-type parasites. In comparison, band sizes of 6.3 and 2.7 or 6.6 and 4.9 kb were detected in marked attB+ recombinant parasites digested with S/E or S/N, respectively. Unmarked NF54attB parasites yielded a single band of 3.3-kb on digestion with either restriction enzyme pair, consistent with loop-out recombination and excision of the selectable markers.

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1112037108 Adjalley et al. Downloaded by guest on September 30, 2021 The genomic organization of an unmarked parasite clone, pfs16-GFP-LUC-attP PNAS PLUS attB ﬁ A named NF54 , was con rmed by Southern blot hybridization pBluescript (Fig. 1C).

P. falciparum Generation of Recombinant Parasites Expressing GFP- attP BSD GFP-LUC Luciferase Under the Control of Gametocyte-Specific Promoters. Gametocyte-specific promoters were chosen from genome-wide expression profiles of the developmentally distinct asexual and NF54attB sexual blood stages (17, 18). We selected pfs16, which is ex- cg6 5’attB cg6 3’ pressed throughout the five stages of gametocytogenesis and is one of the earliest markers of gametocyte development, pfs48/45, BSD selection which is principally active in early and intermediate gametocyte Transgenic GFP-LUC reporter lines fi stages, and mal8p1.16, which is speci c for late stage game- cg6 5’ attL BSD GFP-LUC attR cg6 3’ tocytes (19). Although both pfs48/45 and mal8p1.16 are mini- mally expressed in asexual parasites, pfs16 transcripts can be detected postgametocyte induction in asexual parasites that have B C pfs16 pfs16 promoter activity -GFP-LUC committed to sexual differentiation in the next cycle of intra- 1.2 erythrocytic development (20). These promoters were cloned 1.0 fi fl 0.8 upstream of a GFP- re y luciferase fusion, and the resulting 0.6 attP-containing gametocyte reporter plasmids were cotrans- 0.4 attB 0.2 fected into NF54 parasites along with the pINT plasmid that 0.0 Luciferase Values expresses Bxb1 mycobacteriophage integrase (Fig. 2A). Inte- 02468101214 pfs16 Days of gametocytogenesis grase-mediated attB × attP recombination yielded NF54 , pfs48/45 pfs48/45 mal8p1.16 pfs48/45 promoter activity -GFP-LUC NF54 , and NF54 reporter lines, which were con- 1.2 firmed by PCR and subsequently cloned. 1.0 0.8 To profile sexual stage reporter activity, we triggered game- 0.6 tocytogenesis by starvation-induced stress (21). Luciferase ac- 0.4 0.2

tivity was measured in gametocytes harvested daily for 12–14 0.0 MICROBIOLOGY Luciferase Values 02468101214 consecutive days (Fig. 2B). Expression of the reporter driven by Days of gametocytogenesis the pfs16 promoter began, as anticipated, in asexual parasites mal8p1.16-GFP-LUC ∼ mal8p1.16 promoter activity after gametocyte induction and increased 10-fold to peak 1.2 around day 2 of gametocytogenesis (stages I and II). Promoter 1.0 0.8 activity progressively decreased but reached a plateau at a signal 0.6 level ∼threefold above background in mature gametocytes 0.4 – pfs16 0.2 (days 10 12), suggesting that the NF54 reporter line can be 0.0 Luciferase Values 02468101214 exploited to monitor both early stage and mature gametocytes. DIC GFP-Hoechst Merge Expression of luciferase driven by the pfs48/45 promoter Days of gametocytogenesis peaked, as previously reported (18), between days 3 and 5 of Fig. 2. Engineering and characterization of gametocytogenesis-specific pfs48/45 gametocytogenesis, which makes the NF54 reporter line GFP-luciferase reporter lines. (A) Schematic of the Bxb1 integrase (INT) well suited to monitor stages II and III development. Compari- -mediated genomic insertion of attP-containing plasmids that used game- son of luciferase values between NF54pfs16 and NF54pfs48/45 tocyte-specific promoters (pfs16, pfs48/45,ormal8p1.16) to express the GFP- attB revealed threefold stronger maximal promoter activity for the luciferase (GFP-LUC) fusion. Cloned unmarked NF54 parasites were pfs16 5′ UTR (Fig. 2). Reporter expression under the control of cotransformed with the integrase-expressing plasmid pINT (14) and GFP- LUC-attP plasmids. After integration, the attB site was destroyed, leaving left the mal8p1.16 promoter was minimal in the early stages of fl – (attL) and right (attR) anking regions. (B) Assessment of pfs16, pfs48/45, gametocytogenesis and only increased 6 8 d after commitment and mal8p1.16 promoter-driven luciferase expression throughout game- to sexual differentiation. Promoter activity peaked on days 10–12 tocytogenesis. Luciferase signals were measured daily from triplicate wells of gametocytogenesis, corresponding to expression in late stage harvested from the parasite lines NF54pfs16,NF54pfs48/45, and NF54mal8p1.16. (stages IV and V) gametocytes (Fig. 2B). Fluorescence micros- Values were normalized to gametocyte numbers and plotted as a proportion copy confirmed the expression of the GFP-luciferase fusion in of peak promoter activity (normalized to 1.0; mean ± SEM peak luciferase the gametocyte cytosol for all reporter lines (Fig. 2C). values were 152,398 ± 1,722, 52,052 ± 897, and 2,081 ± 506 for NF54pfs16, NF54pfs48/45, and NF54mal8p1.16). Days of gametocytogenesis are represented Gametocytocidal Activity of Common Antimalarial Drugs. We on the x axis. (C) Assessment of gametocyte promoter-driven GFP expression fl pfs16 pfs48/45 mal8p1.16 assessed the gametocytocidal activity of a panel of nine anti- by uorescence microscopy. NF54 , NF54 , and NF54 parasites were imaged as gametocyte stages V, III, and V, respectively. malarial drugs, including DHA, the active metabolite of artemisinins and itself a first-line agent, and the ACT partner drugs lumefantrine, monodesethyl-amodiaquine (mdAQ; the active against asexual blood stage parasites. IC50 values (Table 1) were metabolite of amodiaquine), piperaquine, and pyronaridine. <70 nM for all compounds tested, with the exception of the pro- Additionally, we tested the Malarone component atovaquone, phylactic antiliver stage agents primaquine and tafenoquine, which the 8-aminoquinolines primaquine and tafenoquine, and the were far less active against asexual stages (IC values of 1.3 and 4.4 thiazine dye MB (structures listed in Fig. S1). Given our em- 50 μM, respectively). Treatment was initiated on day 2 (corresponding phasis on drugs that are used in first-line therapy or are candidate transmission-blocking agents, we did not include the former to stages I and II), 5 (stage III), 8 (stage IV), or 11 (stage V) after gold standard antimalarial drug, chloroquine. Other studies have induction of gametocytogenesis from synchronized parasite cul- clearly established that chloroquine exerts activity on early stage tures (Fig. 3A). Drug treatment was maintained for a total of 3 d gametocytes (6, 10, 13, 22). followed by the removal of drug. Luciferase activity was measured Gametocytes from the NF54pfs16, NF54pfs48/45, and NF54mal8p1.16 daily for 4–12 d after treatment onset. reporter lines were exposed to compounds at concentrations Each antimalarial produced some inhibition of gametocyte corresponding to 0.5×,1×,and5× the mean IC50 value obtained development during early stages (I and II), with the exception of

Adjalley et al. PNAS Early Edition | 3of10 Downloaded by guest on September 30, 2021 Table 1. Activity of antimalarials on P. falciparum gametocyte maturation and transmission Gametocyte maturation assays Gametocyte transmission assays

Asexual blood stage Reduction in Block in

Antimalarial drug mean 1× IC50 (nM) Fold IC50 I and II III IV V Fold IC50 oocysts transmission

Dihydroartemisinin 24 0.5× +++ −−− Dihydroartemisinin 24 1× +++ + + + 1× + − Dihydroartemisinin 24 5× +++ +++ ++ + 5× ++ − Lumefantrine 66 0.5× −−−− Lumefantrine 66 1× −−−−1× ++ − Lumefantrine 66 5× +++ −−− 5× +++ − Monodesethyl-amodiaquine 61 0.5× +++ −−− Monodesethyl-amodiaquine 61 1× +++ −−− 0.5× −− Monodesethyl-amodiaquine 61 5× +++ + −− 2.5× −− Piperaquine 35 0.5× + −−− Piperaquine 35 1× + −−− 1× −− Piperaquine 35 5× +++ −−− 5× −− Pyronaridine 17 0.5× −−−− Pyronaridine 17 1× + −−− 1× −− Pyronaridine 17 5× +++ −−− 5× +++ − Atovaquone 1.5 0.5× −−−− Atovaquone 1.5 1× −−−− Atovaquone 1.5 5× + −−+ Primaquine 1,320 0.5× + −−− Atovaquone 1,320 1× ++ −−− Primaquine 1,320 5× +++ ++ + + Tafenoquine 4,400 0.5× + −−− Tafenoquine 4,400 1× ++ −−− Tafenoquine 4,400 5× ++ + −− Methylene blue 30 0.5× +++ + + − Methylene blue 30 1× +++ ++ + + 0.25× + − Methylene blue 30 5× +++ +++ +++ ++ 1.25× +++ +++

Mean inhibition relative to no drug control: −, <25%; +, 25–50%; ++, 50–75%; +++, >75%. Gametocyte inhibition data for stages I–V were collated from two to ﬁve independent experiments and are displayed in Figs. 3 and 4. Oocyst data were from two experiments and are summarized in Fig. 5 and Tables S1–S3. Atovaquone, primaquine, and tafenoquine were not tested in transmission assays.

atovaquone, which had little, if any, effect at any stage at the low portantly, these assays monitor drug effect over time, allowing nanomolar concentrations tested (Figs. 3 and 4 and Table 1). us to observe delayed inhibition of mid- to late stage game- Drugs were generally less effective against stage III gametocytes, tocytes in the presence of primaquine and tafenoquine (e.g., and most (with notable exceptions discussed below) displayed stages III and IV in Fig. 4 C and D). As discussed later, the lack minimal activity against stages IV or V that are metabolically less of activity observed with primaquine in vitro, despite reports of active (6) (Table 1). its gametocytocidal properties in humans, may be related to the For DHA, we observed rapid and near-complete inhibition of fact that this drug is rapidly metabolized in vivo and that its luciferase expression by immature gametocytes (already evident metabolites are thought to have the more potent transmission- 3 d after the onset of treatment) (Fig. 3B). A similar level of blocking activity. inhibition, albeit with slower kinetics, was observed in stage III MB proved to be, by far, the most effective gametocytocidal gametocytes exposed to the highest drug concentration (5× IC50; agent tested. This compound inhibited stage I and II game- i.e., 120 nM). DHA also exhibited activity against stage IV and V tocytes by >90% at all concentrations (including 0.5×; i.e., 15 gametocytes (Fig. 3B), although the maximal inhibition only nM) (Fig. 4E). Furthermore, potent dose-dependent killing by reached 60% in stage IV gametocytes and 35% in mature stage MB was observed for all gametocytes up to stage IV. Mature V gametocytes at the 5× concentration. stage V gametocytes were strongly affected by the highest con- Among the ACT partner compounds, mdAQ showed a very centration of the drug (5×; i.e., 150 nM), which produced >90% rapid and potent gametocytocidal activity (>75% inhibition inhibition within 6 d of starting treatment. 3 d posttreatment) against the immature stage I and II sexual forms at all concentrations, including 0.5× (i.e., 30 nM) (Fig. Transmission-Blocking Activities of Antimalarial Compounds. First- 3D). For the other ACT partner drugs lumefantrine, piper- line ACT drugs in clinical use (DHA, lumefantrine, mdAQ, and aquine, and pyronaridine, only the 5× concentration (i.e., 330, piperaquine) or clinical development (pyronaridine) were sub- 175, and 85 nM, respectively) produced comparable levels of sequently evaluated for their impact on P. falciparum gametocyte inhibition against stage I and II parasites (Figs. 3 C and E and infectivity to mosquitoes. Studies addressing this important 4A). None of the ACT partner drugs were effective after para- feature of antimalarial action are rare (23), largely because of sites had reached stage III of gametocytogenesis. the difﬁculty of establishing the appropriate infrastructure to We also tested the gametocytocidal activity of primaquine and house P. falciparum-infected mosquitoes. Our experiments were tafenoquine. Both 8-aminoquinolines were moderately effective designed to test whether ACT drug-treated late stage game- against immature gametocytes at all concentrations tested, but tocytes would exhibit reduced infectivity. We also included only the highest drug concentrations (5×; i.e., 6.6 and 22 μM, MB given its potent activity proﬁle against mature gametocytes respectively) had an effect on late stages (Fig. 4 C and D). Im- (Fig. 4E). Parasite development within the mosquito was in-

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1112037108 Adjalley et al. Downloaded by guest on September 30, 2021 A I I-II II III IV V PNAS PLUS

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NAG Magnet purification day 2 Treatment day 2 to day 4

Treatment day 5 to day 7 Treatment day 8 to day 10 Treatment day 11 to day 13

DHA Treatment of stages I-II DHA Treatment of stage III DHA treatment of stage IV DHA treatment of stage V B 2.0 5 x 2.0 5 x 2.0 5 x 2.0 5 x 1 x 1 x 1 x 1 x 1.6 0.5x 1.6 0.5x 1.6 0.5x 1.6 0.5x

1.2 1.2 1.2 1.2

0.8 0.8 0.8 0.8

0.4 0.4 0.4 0.4

0.0 0.0 0.0 0.0 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12

LMF treatment of stages I-II LMF treatment of stage III LMF treatment of stage IV LMF treatment of stage V C 2.0 5 x 2.0 5 x 2.0 5 x 2.0 5 x 1 x 1 x 1 x 1 x 1.6 0.5x 1.6 0.5x 1.6 0.5x 1.6 0.5x

1.2 1.2 1.2 1.2

0.8 0.8 0.8 0.8

0.4 0.4 0.4 0.4 MICROBIOLOGY

0.0 0.0 0.0 0.0 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12

mdAQ treatment of stages I-II mdAQ treatment of stage III mdAQ treatment of stage IV mdAQ treatment of stage V D 2.0 5 x 2.0 5 x 2.0 5 x 2.0 5 x 1 x 1 x 1 x 1 x 1.6 0.5x 1.6 0.5x 1.6 0.5x 1.6 0.5x

1.2 1.2 1.2 1.2

0.8 0.8 0.8 0.8

0.4 0.4 0.4 0.4

0.0 0.0 0.0 0.0 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12

PPQ treatment of stages I-II PPQ treatment of stage III PPQ treatment of stage IV PPQ treatment of stage V E 2.0 5 x 2.0 5 x 2.0 5 x 2.0 5 x 1 x 1 x 1 x 1 x 1.6 0.5x 1.6 0.5x 1.6 0.5x 1.6 0.5x

1.2 1.2 1.2 1.2

0.8 0.8 0.8 0.8

0.4 0.4 0.4 0.4

0.0 0.0 0.0 0.0

Proportion viable gametocytes0 Proportion viable gametocytes2 Proportion viable gametocytes4 Proportion viable gametocytes 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 Days post-treatment Days post-treatment Days post-treatment Days post-treatment

Fig. 3. Activity of the active artemisinin metabolite DHA and ACT partner drugs on gametocyte maturation in vitro. (A) Experimental scheme to assess impact of antimalarials at different stages of gametocyte maturation. After gametocyte induction by limiting nutrients, cultures were treated for 6 d with NAG to eliminate asexual parasites, and gametocytes were magnet-puriﬁed on day 2 postinduction. Drug treatments of 3-d duration were initiated on days 2, 5, 8, or 11 (corresponding to stages I and II, III, IV, and V). Results are shown for (B) DHA, (C) lumefantrine (LMF), (D) mdAQ, and (E) piperaquine (PPQ). Drugs

were tested at 0.5×,1×, and 5× the IC50 concentration that produced a 50% inhibition of growth of asexual blood stage parasites (Table 1). Assays were performed in triplicate on two to ﬁve independent occasions. The gametocytocidal effect was measured relative to the luciferase signal emitted by untreated gametocyte controls cultured in parallel. Drug-speciﬁc effects (represented as means ± SEM) were calculated for up to 12 d after the beginning of treatment at day 0.

vestigated by assessing the production of oocysts. These form After the production of mature gametocytes in vitro, cultures under the insect midgut epithelium after exflagellation of male were exposed to drug on days 12–14. Gametocytes were for- gametocytes to produce microgametes that fuse with female mulated as artificial mosquito blood meals 3 or 4 d later and fed macrogametes and subsequent parasite traversal across the to A. stephensi mosquitoes. Mosquito midguts were dissected 6 peritrophic matrix surrounding the blood meal (24). For the or 7 d after gametocyte ingestion to ascertain the percentage most frequently prescribed ACT (Coartem), which comprises of infected mosquitoes and quantify oocyst production. Control lumefantrine plus an artemisinin derivative (25), we also exam- mosquitoes fed on untreated NF54 gametocytes had an average ined exflagellation of stage V gametocytes as an indicator of of 39 oocysts per midgut (range = 13–67) (Tables S1–S3). This maturity and infectivity (26). average is considerably higher than oocyst numbers seen in

Adjalley et al. PNAS Early Edition | 5of10 Downloaded by guest on September 30, 2021 A PND treatment of stages I-II PND treatment of stage III PND treatment of stage IV PND treatment of stage V 2.0 5 x 2.0 5 x 2.0 5 x 2.0 5 x 1 x 1 x 1 x 1 x 1.6 0.5x 1.6 0.5x 1.6 0.5x 1.6 0.5x

1.2 1.2 1.2 1.2

0.8 0.8 0.8 0.8

0.4 0.4 0.4 0.4

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Proportion viable gametocytes 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 B ATQ treatment of stages I-II ATQ treatment of stage III ATQ treatment of stage IV ATQ treatment of stage V 2.0 5 x 2.0 5 x 2.0 5 x 2.0 5 x 1 x 1 x 1 x 1 x 1.6 0.5x 1.6 0.5x 1.6 0.5x 1.6 0.5x

1.2 1.2 1.2 1.2

0.8 0.8 0.8 0.8

0.4 0.4 0.4 0.4

0.0 0.0 0.0 0.0 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 C PMQ treatment of stages I-II PMQ treatment of stage III PMQ treatment of stage IV PMQ treatment of stage V 2.0 5 x 2.0 5 x 2.0 5 x 2.0 5 x 1 x 1 x 1 x 1 x 1.6 0.5x 1.6 0.5x 1.6 0.5x 1.6 0.5x

1.2 1.2 1.2 1.2

0.8 0.8 0.8 0.8

0.4 0.4 0.4 0.4

0.0 0.0 0.0 0.0 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 D TFQ treatment of stages I-II TFQ treatment of stage III TFQ treatment of stage IV TFQ treatment of stage V 2.0 5 x 2.0 5 x 2.0 5 x 2.0 5 x 1 x 1 x 1 x 1 x 1.6 0.5x 1.6 0.5x 1.6 0.5x 1.6 0.5x

1.2 1.2 1.2 1.2

0.8 0.8 0.8 0.8

0.4 0.4 0.4 0.4

0.0 0.0 0.0 0.0

Proportion viable gametocytes0 Proportion viable gametocytes2 Proportion viable gametocytes 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 E MB treatment of stages I-II MB treatment of stage III MB treatment of stage IV MB treatment of stage V 2.0 5 x 2.0 5 x 2.0 5 x 2.0 5 x 1 x 1 x 1 x 1 x 1.6 0.5x 1.6 0.5x 1.6 0.5x 1.6 0.5x

1.2 1.2 1.2 1.2

0.8 0.8 0.8 0.8

0.4 0.4 0.4 0.4

0.0 0.0 0.0 0.0

Proportion viable gametocytes 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 Days post-treatment Days post-treatment Days post-treatment Days post-treatment

Fig. 4. Identiﬁcation of potent gametocytocidal activity of MB against P. falciparum gametocytes. Compounds were tested against gametocytes at different developmental stages, and results are illustrated as described in Fig. 3. Compounds were (A) pyronaridine (PND), (B) atovaquone (ATQ), (C) primaquine (PMQ),

(D) tafenoquine (TFQ), and (E) MB. Drugs were tested at 5×,1×, and 0.5× the IC50 concentration that produced a 50% inhibition of growth of asexual blood stage parasites (Table 1).

Anopheles mosquitoes fed with gametocytes from patient iso- observed in a rodent malaria model to moderately inhibit oocyst lates, which typically average one or two (27, 28). production directly within mosquitoes (29). This finding is con- Experiments with the partner drugs mdAQ and piperaquine sistent with our observations that lumefantrine reduced P. fal- showed no detectable reduction in oocyst numbers or trans- ciparum oocyst numbers >80% at the 5× concentration (Fig. 5A mission blockade (Fig. 5), consistent with their lack of activity on and Tables S1–S3), despite the lack of an appreciable effect on late stage gametocytes (Fig. 3 D and E). More potent activity was the viability of late stage gametocytes in vitro (Fig. 3C). Initial observed on gametocyte treatment with DHA, which yielded studies using exflagellation to monitor lumefantrine drug action oocyst numbers in infected mosquitoes that were reduced by on stage V gametocytes revealed an average inhibition of 41% ∼25% and 70% at 1× and 5× concentrations, respectively (Fig. and 53% at 1× and 5× concentrations, respectively. In the case of 5A and Tables S1–S3). Initial studies showed that DHA inhibited pyronaridine, this partner drug was ineffective against mature the exflagellation of drug-treated stage V gametocytes by an gametocytes (Fig. 4A), but it substantially inhibited oocyst pro- average of 44% and 92% at 1× and 5× concentrations, reduction and partially blocked transmission (21–27% of mos- spectively (data obtained in two separate experiments that were quitoes fed on pyronaridine-treated gametocytes remained performed in duplicate). Nonetheless, DHA did not potently uninfected) (Fig. 5). Our findings with lumefantrine and pyro- block transmission, because 13% of mosquitoes at most showed naridine suggest downstream effects on parasite development an absence of oocyst infection (Fig. 5B). after the onset of gamete formation within the mosquito vector. It is worth noting that gametocytocidal activity is not the only The most striking impact on transmission was observed in mechanism for transmission blockade. Lumefantrine has been mosquitoes fed on MB-treated gametocytes. At a low concen-

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1112037108 Adjalley et al. Downloaded by guest on September 30, 2021 A the potent clearance of stage V gametocytes. Furthermore, MB PNAS PLUS Experiment 1 Experiment 2 was the only compound that we tested that dramatically reduced 100 the mosquito infection prevalence. These results agree with 75 and extend preliminary clinical observations that MB-based 50 25 treatment regimens reduced the gametocyte carriage rate in 0 treated patients in addition to being effective in clearing asexual -25 blood stage infections (33). Of note, we observed potent game-

Reduction in oocysts (%) -50 DHA LMF mdAQ PPQ PND MB tocytocidal activity at concentrations as low as 30 nM, corre- B 1x 5x 1x 5x 0.5x 2.5x 1x 5x 1x 5x 0.25x 1.25x sponding to 10 ng/mL. This concentration is readily achieved in humans, for which the average Cmax was found to be 3,900 ng/mL 100 Experiment 1 Experiment 2 after administration of a single 500-mg oral dose to adults (with 75 a mean weight of 67 kg) (30). As a reference, combination trials

50 of MB plus amodiaquine or artesunate in children recently used

25 10 mg/kg MB two times daily (34). In humans, MB is used primarily to treat methemoglobinemia, 0 Block in transmission (%) 2+ DHA LMF mdAQ PPQ PND MB a condition whereby the heme ferrous ion (Fe ) is oxidized to 1x 5x 1x 5x 0.5x 2.5x 1x 5x 1x 5x 0.25x 1.25x the ferric state (Fe3+). The reduced form of MB, which acts as an Fig. 5. Transmission-blocking activity of selected antimalarial drugs. P. electron donor, can reverse this oxidation. Against Plasmodium, falciparum NF54 stage V gametocytes were cultured in the presence of drug the mode of action remains enigmatic (36, 37). The hypothesis for 3 d followed by culture in the absence of drug for 3–4 d before feeding that this highly selective, redox-cycling agent interfered with to female A. stephensi mosquitoes. Midguts of blood-fed mosquitoes were glutathione reductase was recently refuted by the demonstration dissected 6–7 d postfeeding, and oocyst numbers were scored. (A) Percent that MB was equally active against P. falciparum parasites lacking reduction in oocyst formation in mosquitoes fed on drug-treated game- this antioxidant enzyme (38). Other evidence suggests that MB tocytes compared with solvent-treated controls (negative reduction equates can interfere with hemozoin formation, which occurs in the to enhanced oocyst numbers). (B) Percent block in transmission in mosqui- Plasmodium digestive vacuole and permits the detoxiﬁcation of toes fed drug-treated gametocytes. This value denotes the percentage of reactive iron heme moieties formed during hemoglobin degra- mosquitoes in which no oocysts were observed after ingestion of drug- treated parasites; all mosquitoes fed on nontreated or mock-treated para- dation (37). Of note, MB is a weak base that could concentrate in

– MICROBIOLOGY sites had oocysts in their midguts. Transmission assays were performed in this highly acidic organelle (with a pH of 5.2 5.5) (39). Such duplicate, with compounds evaluated at the concentrations indicated. Val- a mode of action would be similar to 4-aminoquinolines such as ues were calculated from an average of 20 mosquitoes (range = 14–25) per chloroquine or amodiaquine. However, mechanisms of resistance drug treatment or untreated or DMSO mock-treated control (details pro- to those drugs, mediated by the multidrug transporters PfCRT vided in Tables S1–S3). and PfMDR1 (40, 41), do not impart cross-resistance to MB (36, 42), and a separate mode of action must underlie MB activity × – against mature gametocytes in which hemoglobin degradation no tration (1.25 the IC50, corresponding to 38 nM) (Table 1), 78 longer occurs. Given that these forms are often refractory to 100% of the mosquitoes harbored no oocysts, and the remainder – antimalarial drugs, our data suggest that MB or a derivative harbored a single oocyst, corresponding to a 98 100% reduction thereof could be a highly effective transmission-blocking adjunct in oocyst density compared with controls (Fig. 5 A and B and to curative antimalarial combinations. – Tables S1 S3). This effect is consistent with the substantial de- In response to the global dissemination of P. falciparum strains crease in viable mature gametocytes in MB-treated cultures resistant to the former first-line drugs chloroquine and sulfa- (Fig. 4E). doxine-pyrimethamine, artemisinins have recently been adopted worldwide as the first-line antimalarial drugs, and they are used Discussion in combination with partner drugs that have longer plasma half- fi Here, we report gametocyte stage-speci c transgenic lines that lives (25). Our luciferase data reveal that DHA, the active me- fi allow P. falciparum maturation to be quanti ed throughout the tabolite of artemisinins, has appreciable activity on early stage fi ve stages of gametocytogenesis. These reporter lines with stably gametocytes and to a lesser extent, late stages. Our studies also integrated GFP-luciferase expression cassettes were used to reveal DHA-mediated impairment of both microgametocyte quantitatively assess the activity of several key antimalarials on exflagellation and numbers of oocysts formed within the mos- sexual stage development. In vitro results were complemented by quito. A 70% decrease in oocyst numbers was achieved at con- direct observation of antimalarial drug action on gametocyte centrations (120 nM) that remain biologically relevant (mean transmission to Anopheles mosquitoes. maximal plasma concentration of DHA in a 3-d treatment reg- Our data reveal highly potent gametocytocidal and trans- imen of DHA-lumefantrine is ∼250 nM) (43). Nevertheless, the mission-blocking properties of the thiazine dye MB. Character- block in transmission, measured by the percentage of infected ized by a short plasma half-life and a high bioavailability (18 h mosquitoes, was minimal. This finding is consistent with field- and 72%, respectively) (30), this compound was the first syn- based studies that document a rather modest effect of artemi- thetic compound ever used in clinical therapy, dating back to the sinins on transmission of mature gametocytes (8, 9). A recent 1891 report of its antimalarial properties by the renowned finding that artemisinins inhibit hemoglobin uptake and its sub- chemist Paul Ehrlich (31, 32). Recently, there has been renewed sequent hydrolysis may explain the marked activity of DHA interest in this affordable and already clinically registered com- against immature gametocytes (44). pound, with a series of studies that have assessed the benefitof Lumefantrine, the arylaminoalcohol partner drug of the most adding MB to antimalarial combinations (33–35). One recent widely used ACT Coartem (25), exhibited minimal game- field trial from Burkina Faso observed excellent clinical efficacy tocytocidal potency on sexual forms beyond stage III of differ- of a MB-amodiaquine combination when tested in children with entiation. However, substantial inhibition of infectivity was uncomplicated falciparum malaria (34). Another study reported observed at concentrations (330 nM) far below those levels moderate curative activity with MB monotherapy, illustrating the achieved pharmacologically (mean peak plasma concentration in need for this slow-acting drug to be combined with fast-acting malaria patients was ∼11 μM, with a half-life of 4 or 5 d) (1, 43). agents (35). Our results show that MB interferes with gametocyte The reduction in oocyst numbers exceeded drug impact on development at all stages and can block transmission through exflagellation, suggesting that lumefantrine may impact the ac-

Adjalley et al. PNAS Early Edition | 7of10 Downloaded by guest on September 30, 2021 tivation of female gametocytes, gamete formation, or possibly, Whether these compounds act on gametocytes by inhibiting oocyst development through an unknown mode of action, which mitochondrial function and/or triggering oxidative stress remains was suggested by recent findings from rodent models (29). to be elucidated (49). Primaquine, however, has known toxicity Amodiaquine in combination with artesunate constitutes the in G6PD-deficient patients, primarily as a result of hemolytic second most widely used ACT, and the related bisquinoline anemia (56). These safety concerns, combined with the elevated piperaquine has recently become a first-line drug in some prevalence of this genetic disorder in malaria-endemic pop- Southeast Asian countries. Pyronaridine, a Mannich base, has ulations (57), emphasize the need to pursue alternative 8-ami- completed Phase III clinical trials in combination with artesu- noquinolines with reduced hematoxicity and also explore alter- nate, and this new ACT is under review for registration (1, 25). native chemical scaffolds that block the transmission of mature The chemical similarity of mdAQ, piperaquine, and pyronaridine gametocytes. to chloroquine, a 4-aminoquinoline, suggests a related mode of Overall, our results suggest that, of the current ACTs, com- action involving inhibition of heme detoxification in the digestive binations pairing an artemisinin derivative with lumefantrine vacuole (39). Consistent with this activity, mdAQ, piperaquine, or pyronaridine might be the most effective at targeting both and pyronaridine exert gametocytocidal activity only on imma- asexual and sexual stages. Conversely, the partner drugs piper- ture gametocytes. Both mdAQ and piperaquine treatments aquine and mdAQ revealed no notable transmission-blocking failed to modulate gametocyte transmissibility when treating activity, which was measured through oocyst density and in- mature gametocytes. In contrast, pyronaridine (which is char- fection prevalence in mosquitoes. Furthermore, none of the acterized by a relatively long terminal half-life of 7–9 d) treat- current ACT partner drugs were effective against mature stage V ment led to a significant reduction in oocyst numbers at con- gametocytes, which was quantified in our luciferase assays, po- centrations still below the mean maximum plasma concentration tentially reflecting the less metabolically active nature of late in patients (160 nM) (45). Thus, although pyronaridine does not stage gametocytes that renders them insensitive to many anti- seem to affect gametocyte viability per se, as measured by ex- malarials. Finally, we find that the broadly active compound MB pression of luciferase, our evidence suggests inhibition of sub- might be an excellent gametocytocidal drug to add to the ACT sequent parasite development within the mosquito. armamentarium. Primaquine has a long history of use for the treatment of P. Our results with known antimalarials highlight the use of these vivax and P. ovale malaria, where it is effective at preventing GFP-luciferase reporter lines for deriving a quantitative readout relapses through its activity against liver stage hypnozoites (7). of P. falciparum viability throughout gametocyte development. Studies with P. falciparum infections indicate that a single dose This system allows for the precise determination of both drug of primaquine supplemented to ACTs can substantially shorten potency and kinetics of inhibition, revealing the dose-dependent the period of gametocyte carriage and importantly, clear sub- susceptibility of each gametocyte stage. Both the luciferase and microscopic gametocytes (46, 47). In our assays, this 8-amino- GFP stably integrated reporters are amenable to high-through- quinoline drug displayed gametocytocidal activity against stage put approaches to screen for gametocytocidal compounds. Fur- I–IV sexual forms, albeit at low micromolar concentrations thermore, NF54 parasites are efficiently transmitted to mos- compared with low to mid-nanomolar concentrations with DHA quitoes, allowing the measurement of drug susceptibilities and MB. Primaquine also exhibited partial activity against ma- during the various steps in the parasite transition from human to ture (stage V) gametocytes, although this finding was only ob- mosquito host. This dual approach of quantitatively assessing served at the 5× concentration that corresponds to 6.6 μM. gametocyte maturation combined with mosquito transmission Pharmacokinetic studies in humans administered a standard oral studies on active compounds can now be leveraged to identify dose of 45 mg primaquine showed Cmax levels of 0.3 and 3.1 μM gametocytocidal agents, with the ultimate objective of designing of the parent compound and its primary metabolite carbox- therapies that both cure malaria and prevent its transmission. yprimaquine, respectively (48). Our data suggest that the rapidly metabolized parent compound is not a potent gametocytocidal Materials and Methods agent and that its transmission-blocking activity observed clini- Plasmid Constructs and Parasite Transfections. To generate the pCC-cg6-attB ′ ′ fi cally may be attributable to metabolites (49). plasmid, 0.4-kb 5 and 3 fragments of cg6 (PlasmoDB identi er PF07_0036) were amplified using primers p1513 + p1514 and p1515 + p1516, respectively We also examined the gametocytocidal potency of the clinical (Table S4). These fragments introduced 5′ and 3′ AatII sites and an internal candidate tafenoquine, a 5-phenoxy derivative of primaquine 44-bp attB sequence (14), and they were used for overlapping PCR using that is less toxic and has a longer plasma half-life (14 d vs. 5–6h, primers p1513 + p1516. The corresponding product was subcloned into respectively) (48, 50). In our assays, tafenoquine was comparable pCR2.1 (Invitrogen), and sequence-verified inserts were excised by AatII and with primaquine in showing gametocytocidal activity (primarily ligated into pCC (58). pCC-cg6-attB was electroporated into NF54 parasites against early stages), albeit at high concentrations (2.2–22 μM) propagated in malaria culture medium (14) supplemented with 10% human (Fig. 4D). These concentrations are equivalent to or exceed the AB+ serum. Transformed parasites were selected using 2.5 nM WR99210, and 1 μM5-fluorocytosine was used to select for excision of the selectable Cmax value of 4 μM observed in human volunteers administered markers after integration. a single oral dose of 400 mg tafenoquine (50). Our data are fi consistent with earlier reports of its limited gametocytocidal Gametocyte stage-speci c reporter vectors were constructed by amplify- ing the 5′ UTRs of the gametocyte genes pfs16 (PFD0310w; 1.3-kb), pfs48/45 activity against rodent P. berghei or P. yoelii parasites, contrasting (PF13_0247; 1.4-kb), and mal8p1.16 (MAL8P1.16; 1.7-kb) with primers (p2145 with the more potent in vivo transmission-blocking action of + p2146, p2147 + p2148, and p2151 + p2152, respectively) containing ApaI or primaquine (51, 52). Studies with rat liver microsomes show that AvrII restriction sites (Table S4). Promoter regions were inserted into ApaI/ tafenoquine is slowly but extensively metabolized (53). However, AvrII-digested pDC2-cam-mRFP-Vps4-attP derived from pLN (14). This plas- we could find no reports of whether these metabolites are mid also contains the Mycobacterium smegmatis attP site and a blasticidin gametocytocidal. Tafenoquine has been reported to inhibit S-deaminase selectable marker cassette. The GFPmut3/LucIAV fusion cassette sporogony (i.e., the formation of sporozoites inside oocysts) in was isolated from the pU.K.GFPM3lucIAVcl1 plasmid (provided by B. Franke- P. falciparum, P. vivax, and P. berghei (54, 55) and thus, has been Fayard and C. Janse, Leiden, The Netherlands) after XbaI and XhoI digestion. described as transmission-blocking by virtue of this sporonticidal This GFP-luciferase fusion was cloned downstream of the gametocyte promoters and upstream of an hsp86 3′ UTR using AvrII and XhoI sites. action (absent with the more potent gametocytocidal drug The resulting plasmids (pfs16-GFP-LUC-attP, pfs48/45-GFP-LUC-attP, and primaquine). Additional experiments with NF54 parasites are mal8p1.16-GFP-LUC-attP) were cotransfected with the Bxb1 mycobacter- planned to investigate dose-dependent effects of tafenoquine and iophage integrase-expressing vector pINT into NF54attB parasites as de- primaquine on gametocyte transmission as well as sporogony. scribed (14). Transfectants were selected with 2 μg/mL blasticidin (Invitrogen)

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1112037108 Adjalley et al. Downloaded by guest on September 30, 2021 and 250 μg/mL G418 (for the first 6 d only), and they were first detected 20 d Gametocytes were transferred into 96-well plates at 2% parasitemia PNAS PLUS postelectroporation. Plasmid integration was confirmed by PCR, and clones and 2% hematocrit starting on day 2 postinduction and cultured in the were obtained by limiting dilution. presence of NAG. Drugs were added for 3 d, with treatment initiated on days 2 (stages I and II), 5 (stage III), 8 (stage IV), or 11 (stage V) of gametocyto- DNA Analysis. P. falciparum trophozoite-infected erythrocytes were har- genesis, with daily medium changes. Stage I and II, III and IV, and V game- vested and saponin-lysed. Parasite genomic DNA was extracted and purified tocytes were assessed using the pfs16-GFP-LUC, pfs48/45-GFP-LUC, and both using DNeasy Tissue kits (Qiagen). Integration of pCC-cg6-attB into the cg6 pfs16-GFP-LUC and mal8p1.16-GFP-LUC lines, respectively. Untreated locus of NF54 parasites was detected using primers p1655 + p838 (specificto gametocytes, maintained on the same culture plates and processed in par- the cg6 5′UTR and the PbDT 3′UTR, respectively) and primers p1656 + p1636 allel, were used as controls. (specifictothecg6 3′ UTR and human dhfr, respectively). These primer pairs To quantify the inhibitory effects of drugs on gametocytes, luciferase produced bands of 3.4 and 2.7 kb, respectively, in integration-positive par- counts were normalized based on a weighted average of the data. The first asites. Subsequent intralocus recombination, leading to excision of the se- part of the weighting consisted of dividing the luciferase counts for each day lectable markers, was identified using primers p1969 + p1970, both specific of the assay for treated and control lines by their starting values (day 0 or 1). to cg6 (Table S4). PCR amplification with p1969 + p1970 yielded a doublet Values for drug-treated parasites were then divided by values obtained for band migrating around 0.2-kb corresponding to fragments containing or nontreated controls. The second part of the weighting consisted of dividing lacking an internal attB site (depending on whether recombination leading the raw treated values by the raw control values for each day of treatment. ′ to loss of the human dhfr and cdup markers occurred between the cg6 5 or The average of these two calculations was then plotted as a function of time ′ 3 sequences) (Fig. 1A). The presence of attB in the unmarked cg6 locus was (Figs. 3 and 4). This method standardized the assays to a common scale while fi fi con rmed using primers p1829 (speci c to the attB site) and p1656, yielding separating drug action from background changes in promoter activity or × fi a 0.8-kb band. Integrase-mediated attB attP site-speci c recombination gametocyte numbers over time. was detected using primers located on each side of the integration: p1969 + fi ′ p836 (speci cforcg6 and PcDT 5 UTR, respectively) and p1970 + p984 Transmission-Blocking Studies and Oocyst Formation. NF54 gametocytes were (specificforcg6 and the firefly luciferase coding sequence, respectively). induced by allowing asexual blood stage parasites to proliferate without the For Southern blot analysis, 1 μg genomic DNA was digested with SalI/NruI addition of fresh erythrocytes (60). Gametocyte cultures in six-well plates or SalI/EcoRV enzymes, electrophoresed on a 0.7% agarose gel, and trans- were treated at 1× or 5× the mean IC value (obtained against asexual ferred onto a Nytran nylon membrane overnight. Hybridization was per- 50 blood stages) for all drugs except mdAQ (0.5× and 2.5×) and MB (0.25× and formed at 55 °C with a 0.4-kb [32P]-cg6 probe obtained as a SalI/AatII 1.25×). All cultures were prepared in parallel with controls corresponding to fragment from pCC-cg6-attB. cultures that were untreated or treated with 0.05–0.1% DMSO, except for MB (water control). Gametocytes were exposed to drug with daily media Gametocyte Induction and Purification. To produce gametocytes for in vitro changes from days 12 to 14 after their induction. Parasites were sub- assays, asexual blood stage parasites were first synchronized by 5% D-sorbitol MICROBIOLOGY sequently maintained in the absence of drug until blood feeding on days 17 (wt/vol) treatment for 10 min at 37 °C for two or more successive growth cycles. and 18. The percentages of mature gametocytes in cultures were assessed Synchronous parasites at 3% hematocrit were cultured in 100-cm2 Petri dishes by microscopy of Giemsa-stained thin blood smears. Functional micro- to 10% parasitemia (ring stage), at which point gametocytogenesis was in- gametocytes were assessed for exflagellation activity at 18 °C after drug duced by feeding cultures with 50% conditioned media (21). The next day, treatment of stage V gametocytes on days 15–17 and were quantified as trophozoite cultures were diluted fourfold. N-acetyl glucosamine (NAG; exflagellation centers per 106 human erythrocytes. Sigma-Aldrich) was then added to a final concentration of 50 mM after all Treated and control cultures were formulated as artificial mosquito blood schizonts had ruptured. NAG treatment was maintained for the next two to fi – three cycles of reinvasion to eliminate all asexual forms from the culture. Im- meals at 50% hematocrit, with nal stage V gametocytemias of 0.01 0.1% mature gametocytes (stages I and II) were magnetically purified by passage (after dilution of the starting cultures in which gametocytemias typically – through a MACS CS column (Miltenyi Biotech) and transferred into 96-well reached 0.7 1.7%). Blood meals were fed in duplicate to A. stephensi using fi plates at 2% hematocrit such that each well contained 1–2% gametocytes in arti cial membrane feeders. Engorged mosquitoes were maintained at 26 °C a final volume of 150 μL. Giemsa-stained smears were examined daily to and 78% relative humidity with 12-h light/dark cycles. In each feeding ex- monitor development. periment, between 15 and 25 mosquitoes with developed ovaries (indicating blood feeding) were sampled 6 or 7 d postfeeding to assess oocyst formation. Mosquito midguts were dissected in 0.2% Mercurochrome, and oocyst Luciferase Assays. Gametocytes were harvested daily for up to 12–16 d. Cultures were concentrated by centrifugation, and pellets were stored at numbers were counted under a phase-contrast microscope. The infection fi −80 °C. Cells were lysed at room temperature in 3× Luciferin Lysis Buffer prevalence (de ned as the percentage of mosquitoes with one or more oocyst) was determined for each experiment. Routinely, this prevalence was (15 mM DTT, 0.6 mM CoA, 0.45 mM ATP, 0.42 mg/mL luciferin, 10 mM MgCl2, 10 mM Tris Base, 10 mM Tris·HCl, 0.03% Triton X-100). Luciferase activity was 100% in mosquitoes fed untreated or mock-treated (DMSO) gametocytes – measured at 560 nm in a Victor3 luminescence plate reader (Perkin-Elmer). (Tables S1 S3). The intensity of mosquito infection was also evaluated as the GFP expression from the different gametocyte-specific promoters was con- mean number of oocysts per midgut. A Student t test was performed to fi firmed by fluorescence microscopy using a Nikon TiE PFS inverted micro- determine statistical signi cance. scope equipped with a CoolSNAP HQ2 monochrome camera. SI Materials and Methods Details. In SI Materials and Methods, Fig. S1 shows – Drugs and in Vitro Susceptibility Assays. Atovaquone (provided by A. Vaidya, chemical structures, Tables S1 S3 summarize compound activity against Philadelphia, PA), DHA (Avachem Scientific), lumefantrine (a gift from Philip P. falciparum gametocyte transmission to Anopheles mosquitoes, Table S4 Rosenthal, San Francisco, CA), MB (American Regent), mdAQ (a gift from lists oligonucleotide sequences, and Table S5 shows the raw data used to Pascal Ringwald, Geneva, Switzerland), piperaquine (Avachem Scientific), generate Fig. P1 in the Author Summary. primaquine (Sigma), pyronaridine (Avachem Scientific), and tafenoquine (GlaxoSmithkline) were used at three different concentrations corresponding ACKNOWLEDGMENTS. We thank Joerg-Peter Kleim and others at Glaxo- SmithKline for providing tafenoquine and Neha Passi and Catie Brownback to 0.5×,1×, and 5× the mean IC50 value obtained with asexual blood stage parasites (Table 1). In vitro IC values against asexual parasites were calcu- for assistance with the manuscript. G.L.J. received funding through the 50 National Science Foundation Graduate Research Fellowship Program. lated by nonlinear regression analysis of 72-h [3H]hypoxanthine-based growth Funding support for R.T.E. was provided by US National Institutes of Health inhibition assays (59). All drugs were prepared as stock solutions in 100% Grant T90 NR010824. This work was supported in part by US National DMSO except for MB, which was dissolved in water. Drugs were diluted in Institutes of Health Grant AI079709 (to D.A.F.) and the Medicines for × RPMI-1640 to 50 IC50 (DHA was prepared fresh because of its poor stability in Malaria Venture (D.A.F.). Sanaria Inc. provided funding for the transmission- aqueous solution). blocking experiments.

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